Traditional chemical processes can be harmful to the environment. Enzymatic bioprocessing, on the other hand, uses enzymes to catalyze chemical reactions, and therefore, represents an important approach in developing environmentally safe processes for applications such as bulk chemical production and drug synthesis. Enzymatic bioprocessing can be used in producing chemical intermediates such as alkene epoxides and glycosides; the former are among the top 50 commodity chemicals in the United States and the latter are commonly used in drug synthesis. Additional enzymatic bioprocessing applications include oxidizing organic pollutants such as polycyclic aromatic hydrocarbons and enzymatically desulfurizing and refining petroleum.
Unfortunately, the prior art has failed to yield an efficient means for performing enzymatic bioprocessing partly because of an enzyme's inherent hydrophilicity. Many significant enzymatically-catalyzed reactions involve both hydrophilic and hydrophobic reactants dissolved in polar and nonpolar solvents respectively. The unmodified catalytic enzymes, which hereinafter may be referred to as native enzymes, have a hydrophilic nature that only allows them to effectively dissolve in polar solvents, e.g., water. Therefore, their very nature limits their accessibility to reactants in a nonpolar solvent.
When hydrophilic and hydrophobic reactants are dissolved in separate immiscible solvents, enzymatic catalysis and chemical reaction can only occur at the interface created where the polar and nonpolar liquids are in contact. But the bulk of the reactants and enzymes in solution rarely reach the biphasic interface due to their individual kinetics, and when they do, it is only momentary. With respect to all of the native enzymes in solution, only a very small number are available at the biphasic interface at any point in time. The unavailability of reactants and enzymatic catalysts at the interface, i.e., reaction site, significantly contributes to the bioprocess's overall inefficiency. Thus, the reaction rates of these traditional enzymatic bioprocesses are slow in comparison to those of traditional chemical processes.
There are exceptions to the general rule regarding an enzyme's hydrophilicity. Certain proteins can bind to lipid membranes via Coulombic force, Born repulsion, and hydrophobic interactions; with electrostatic interactions between the positively charged protein domain and the negatively charged lipid membrane being considered the key driving force in most cases. Lipases, which hydrolyze triacylglycerol lipids, are a unique class of enzymes that assemble at a lipid-water interface through hydrophobic interactions. It has been revealed that pancreatic lipase assembles at a lipid-water interface via complexation with pancreatic colipase, which provides the necessary hydrophobicity. Other lipases such as Rhizomucor miehei lipase have surfaces with sufficient hydrophobicity to enable assembling at a lipid-water interface. Enzymes other than lipases generally lack the hydrophobicity for assembly at organic-aqueous interfaces.
Attempts have been made to manipulate the hydrophilic nature of enzymes in order to make them more useful in nonpolar mediums. For example, V. M. Paradar and J. S. Dordick (J. Am. Chem. Soc., 1994, vol. 116, 5009) demonstrated that enzymes can be ion-paired with surfactants and thereby form enzyme-surfactant ion-paired complexes; they can be formed by contacting an aqueous enzymatic solution with a nonpolar solution comprising surfactants such as AOT. Upon contact, electrostatic interactions cause the surfactants to pair with the enzymes, and the resulting enzyme-surfactant complex is soluble in nonpolar solvents. These complexes have been found particularly useful as enzymatic catalysts in reactions occurring in nonpolar solvents. However, the complexes are not useful in biphasic polar-nonpolar liquid reaction systems.
Similarly, other attempts to manipulate the lipophobicity of enzymes also include chemical modification, such as attaching polyethylene glycol (PEG) (for example, see C. Pina, D. Clark, H. Blanche and I. G. Gonegani, Biotechnology Techniques, 2989, vol. 3, 333; P. Wang, C. A. Woodward, E. N. Kaufman, Biotechno. Bioeng., 1999, vol. 64, 290; Z. Yang, Progress in Biochemistry and Biophysics, 1995, vol. 22, 340). Examples of further attempts include deglycosylation followed by attaching benzyl groups (see R. Vazquez-Duhalt, P. M. Fedorak, Enz. Micro. Tech. 1992, vol. 14, 837). Again, these manipulations all result in modified enzymes that are suitable for use in monophasic systems such as polar or nonpolar solvents, but not the immiscible biphasic systems addressed with this invention.